Inflammatory as well as normal physiological processes produce a host of harmful oxidizing substances such as reactive oxygen species (ROS), reactive nitrogen species (RNS), reactive electrophilic metabolites, and lipid peroxidation products. To protect against these harmful substances, the human body has developed intrinsic antioxidant response mechanisms to upregulate a number of antioxidative and cytoprotective enzymes that include glutathione S-transferases (GST), UDP-glucuronyl transferase 1A1 (UGT1A1), NAD(P)H:quinone oxidoreductase 1 (NQO1), catalase, superoxide dismutase 1 (SOD1), and heme oxygenase 1 (HO-1). There are three major cellular components involved in the regulation of antioxidant response; they are Kelch-like ECH-associated protein 1 (Keap1), nuclear factor erythroid 2-related factor 2 (Nrf2), and antioxidant response elements (ARE). The Keap1-Nrf2-ARE is the main signaling pathway that regulates a series of cytoprotective proteins at the transcriptional level including Nrf2 itself as shown in FIG. 1. This signaling pathway induces an adaptive response for oxidative stress which can otherwise lead to many inflammatory diseases. In fact, inflammation has been recognized as an underlying contributor to virtually every chronic disease. Thus, targeting the Keap1-Nrf2-ARE signaling pathway is an attractive strategy to discover preventive and therapeutic agents as antioxidant inflammation modulators (AIMs) for diseases and conditions, including cancer, diabetes, Alzheimer's, and Parkinson's, that involve oxidative stress and inflammation.
Some of the known Nrf2-ARE inducing agents are already in human clinical trials as chemopreventive agents for cancer or as therapeutic agents for conditions involving inflammation. For example, sulforaphane, an isothiocyanate found in cruciferous vegetables and a known ARE inducer, is being tested in clinical trials for the treatment and prevention of prostate cancer and for the treatment of chronic obstructive pulmonary disease (COPD). Bardoxolone methyl, another potent inducer of the Nrf2 pathway, was tested in phase III clinical trials as a first-in-class AIM for the treatment of advanced chronic kidney disease (CKD) in patients with type 2 diabetes mellitus. However, all currently known small molecule Nrf2/ARE inducers are believed to be irreversible modifying agents of cysteine sulfhydryl groups and these include many natural products (e.g., sulforaphane, curcumin, and epigallocatechin gallate from natural sources such as fruits, vegetables, and tea products) and synthetic compounds (e.g., oltipraz, anethole dithiolethione, bardoxolone methyl). All these compounds are either chemically reactive or can be converted to chemically reactive metabolites that readily oxidize or form covalent adduct with the sulfhydryl group of cysteines. It is believed that this modification of cysteine residues in Keap1 is responsible for the disruption of Keap1-Nrf2 complex formation and degradation, leading to subsequent translocation of Nrf2 into the nucleus. Thus, these known modulators are all considered indirect irreversible inhibitors of Keap1-Nrf2 interaction. The reactivity of these compounds raises safety concerns over their long-term use as chemopreventive and therapeutic agents. Development as such is questionable because of concerns about their long-term toxicity. Indeed, bardoxolone methyl was recently withdrawn from Phase III clinical trials citing safety concerns over adverse events and increased rate of death.
Direct disruption of Keap1-Nrf2 protein-protein interaction using small molecule inhibitors, though more challenging, represents an attractive novel strategy to promote translocation of Nrf2 to the nucleus and elevate the expression of ARE enzymes. The only direct inhibitors of Keap1-Nrf2 interaction currently known are the peptides based on the Nrf2 Neh domain that are used in the crystallographic studies. Multiple charges are present on these peptides and their poor membrane permeability and susceptibility to proteolysis prevented their use directly in cellular and in vivo assays. Therefore, there is an urgent need to discover potent small molecule direct inhibitors of Keap1-Nrf2 protein-protein interaction as proposed in this application.